Serotype and multilocus sequence typing of Streptococcus suis from diseased pigs in Taiwan

Streptococcus suis (S. suis) infection can cause clinically severe meningitis, arthritis, pneumonia and septicemia in pigs. To date, studies on the serotypes, genotypes and antimicrobial susceptibility of S. suis in affected pigs in Taiwan are rare. In this study, we comprehensively characterized 388 S. suis isolates from 355 diseased pigs in Taiwan. The most prevalent serotypes of S. suis were serotypes 3, 7 and 8. Multilocus sequence typing (MLST) revealed 22 novel sequence types (STs) including ST1831-1852 and one new clonal complex (CC), CC1832. The identified genotypes mainly belonged to ST27, ST94 and ST1831, and CC27 and CC1832 were the main clusters. These clinical isolates were highly susceptible to ceftiofur, cefazolin, trimethoprim/sulfamethoxazole and gentamicin. The bacteria were prone to be isolated from cerebrospinal fluid and synovial fluid in suckling pigs with the majority belonging to serotype 1 and ST1. In contrast, ST28 strains that corresponded to serotypes 2 and 1/2 were more likely to exist in the lungs of growing-finishing pigs, which posted a higher risk for food safety and public health. This study provided the genetic characterization, serotyping and the most current epidemiological features of S. suis in Taiwan, which should afford a better preventative and treatment strategy of S. suis infection in pigs of different production stages.

MLST. Seven house-keeping genes, including aroA, cpn60, dpr, gki, mutS, recA and thrA, were amplified by PCR for the nucleic acid extracted from the purified S. suis isolates according to the MLST method established by King et al. and Rehm et al. 3,17 . The confirmed PCR products were then sent to Tri-I Biotech, Inc. (Taiwan) for 5' to 3' and 3' to 5' sequencing using an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, USA). Subsequently, the sequencing results were uploaded to BioNumerics® version7.6.3 (Applied Maths, USA), and the obtained alleles and STs were compared with the PubMLST database. New allele sequences and STs were uploaded to PubMLST to obtain the allele profile and define the genotypes 18 . By using the eBURST analysis of PubMLST, the S. suis isolates with respective allele profiles were clustered according to the association of STs. When 6 of 7 alleles among the S. suis isolates were identical, these isolates were identified as the same cluster. The isolates that did not belong to any cluster were singletons 3 . The minimum spanning tree (MST) was calculated using BioNumerics® version 7.6 according to the unweighted pairgroup method with an arithmetic mean algorithm (UPGMA). In order to understand the general distribution of the S. suis isolates, MST was set as distance ≤ 1 for partitioning, in which the involving nodes were clustered as CCs 19 .
Statistical analysis. Association of isolation sites, serotypes, and antimicrobial drug resistance were analyzed by Chi-square test and Fisher's exact test, depending on the number of samples. SPSS was used for statistical analysis and p value < 0.05 was considered to be statistically significant. If p value < 0.01, there was an extremely significant association.  , five pigs had one strain with an unknown serotype; and for the other strain,  there were 3 pigs with serotypes 4, 7, and 8 individually and 2 pigs with serotype 9. The remaining two of the seven pigs were serotypes 3 and 8, and serotypes 7 and 8, respectively. Serotype 1 was frequently isolated from brains, cerebrospinal fluid or synovial fluid at the suckling stage (p < 0.01). Serotypes 3, 7, 8, and 9 were mostly isolated from lungs or bronchial lumina at the nursing stage. In addition, serotype 2 was most likely isolated from lungs at the growing-finishing stage (p < 0.05) ( Table 1).
The relation between the antimicrobial susceptibility and the serotypes indicated that serotype 1 was highly susceptible to penicillin G, amoxicillin, cefazolin, ceftiofur, tiamulin and enrofloxacin. Serotypes 2 and 3 also had high susceptibility to penicillin G, amoxicillin, cefazolin, ceftiofur, enrofloxacin, tiamulin; and were additionally highly susceptible to gentamicin, florfenciol and trimethoprim/sulfamethoxazole. Serotype 7 had better susceptibility only to cefazolin, ceftiofur and trimethoprim/sulfamethoxazole. Serotypes 8 and 9 were highly susceptible to cefazolin, ceftiofur, gentamicin and trimethoprim/sulfamethoxazole (Table S1).

Discussion
In this study, S. suis isolates were mostly serotypes 3, 7 and 8, followed by serotypes 1, 2 and 9. In comparison with the serotype distribution in other countries, serotype 2 is the most prevalent in China, Japan, Vietnam, Thailand, Spain, Italy, France, Poland and White Russia 20 . The second prevalent serotype varies with countries, which are mostly serotypes 3 and 4 in Korea, serotypes 3, 1/2 and 7 in the U.S., serotype 9 in the Netherlands, and serotypes 1 and 14 in Britain 21,22 . Our result is close to the data reported in the U.S., in which serotypes 3 and 7 are more prevalent. In addition, our results are also in agreement with previous finding that S. suis isolated from diseased pigs was mainly composed of limited serotypes 23 . In general, the cps genome is the key region of gene recombination, leading to cps transformation between strains 24 . New cps genomes of S. suis have been found successively in recent years 25 , which expands the genetic diversity and reduces the quantity of unidentified isolates. In the current study, there are 18.8% of isolates that cannot be identified using cps gene by PCR and RFLP; therefore, evaluation of the virulence potential and the effects of these likely new genomes in the pathogenesis is warranted.
As shown in Table 1, serotype 1 was frequently isolated from brains, cerebrospinal fluid or synovial fluid at the suckling stage (p < 0.01). Furthermore, a very significant association between the stage of suckling pigs and the strains derived from cerebrospinal fluid and synovial fluid was found (p < 0.001, Table S2). The phenomenon that suckling pigs were more prone to brain and joint infections may be attributed to the invasive operations such as ear notching, tail cutting, teeth clipping, castration and drug injection during the lactation stage, leading to exposure of opportunistic bacteria in the environment such as S. suis. In addition, significant association between the strains isolated from respiratory tract and the stages of growing and finishing pigs was identified. S. suis that colonizes in healthy adult pigs rarely causes symptoms; however, suckling pigs may be prone to get infected from the healthy adult S. suis carriers through cross-fostering 26 .  www.nature.com/scientificreports/ The antimicrobial susceptibility testing showed that the isolates were highly susceptible to ceftiofur, cefazolin, trimethoprim/sulfamethoxazole and gentamicin, and moderately susceptible to florfenicol, amoxicillin, enrofloxacin, tiamulin, penicillin G and doxycycline. By applying the PK/PD indices that are calculated by the antimicrobial susceptibility data (Fig. S1) in combination with pharmacokinetic parameters of the drugs, effective treatment can be reasonably recommended. For instance, the suckling pigs were prone to be infected with serotype 1 which mainly causes suppurative meningitis or/and arthritis, so amoxicillin, ceftiofur and tiamulin were recommended for treatment. The pigs at the nursing stage were especially susceptible to serotypes 3, 7, 8 and 9, in which the infection in the respiratory system was relatively frequent. Considering the pharmacokinetic characteristics, cefazolin, ceftiofur, or trimethoprim/sulfamethoxazole was recommended for treatment. Among them, β-lactams are time-dependent bactericidal antimicrobial agents. In this study, the MIC 90 of S. suis serotype 3, 7, 8 and 9 for ceftiofur were 0.25, 2, 0.5, and 0.25 μg/mL, respectively. After administration of ceftiofur at 5 mg/ (a) Clonal complex (CC) (b) Serotype The relationship between serotypes and STs.  30 . In addition, it should be noted that healthy asymptomatic pigs are the source of human S. suis infection, in which most of the strains are serotype 2 and 90.2% of the cases are in Asia. Therefore, more attention should be paid to S. suis serotype 2 strains persisting in finishing pigs 31 .
The proportion of S suis resistant to macrolides and lincosamides is rising globally 20 . According to the results of the drug resistance the results of the drug resistance, it can be seen that 89.9% of S. suis isolates were resistant to three classes of drugs including tetracyclines, macrolides and lincosamides. Li et al. have reported that S. suis resistant to chloramphenicols, macrolides, lincosamides, chloramphenicols, fluoroquinolones, aminoglycosides and hydantoins is the most common drug resistance profile in China 40 . In addition, S. suis strains become resistant to five classes of antimicrobials including tetracyclines, lincosamides, fluoroquinolones, sulfonamides and hydantoins in Brazil 33 . The percentage of multi-drug resistance (resistance to more than 3 classes of antimicrobials) of the isolates collected in this study was as high as 93.0%, and 39.4% of the isolates were resistant to more than 6 classes of antimicrobials. Our result is similar to the data from Li et al. (2012) and Soares et al. (2014), which showed the proportion of S. suis isolates resistant to more than 3 classes (98.7%, 99.6%) and 6 classes (35.9%, 85.0%) of antimicrobials in China and Brazil 33,40 .
Comparing the MLST result with serotypes, it was apparent that the pigs at the suckling stage were mainly infected with S. suis serotype 1, which corresponded to ST1 (n = 5) and CC1. S. suis serotype 2 mainly corresponded to ST28 (n = 6) ( Table 3). Goyette-Desjardins et al. found that most of the invasive strains with high virulence belong to CC1, including ST1, ST6, ST7 and ST11, which are usually related to septicemia, meningitis and arthritis 21 . The current study showed that S. suis isolated from the diseased pigs with central nervous system symptoms and arthritis also belonged to CC1. In contrast, CC27 might be related more to the respiratory tract infections 3 . S. suis serotype 2 isolated from the growing-finishing pigs was ST28 belonging to CC27 and mostly isolated from the respiratory system. This result is similar to that of China, Japan, and the U.S., but the pathogenicity needs further investigation 21,41 . S. suis serotype 3 isolated from the pigs at the nursing stage was mainly ST27 of CC27 (n = 12) from respiratory systems (79.7%; 63/79). In comparison, S. suis serotype 8 was prone to be isolated from the pigs at the nursing stage (p < 0.01), and it was mostly isolated from lungs or bronchial lumina (82.2%; 37/45). These isolates were mainly ST1831 of CC1832 (n = 5). These results correspond well to previous findings that serotypes 3 and 8 are mostly restricted to lung infection 42 . CC94 included ST94 (n = 11) and ST1175 (n = 2), which corresponded to the unidentified S. suis isolates (n = 6), serotypes 4 (n = 3), 7 (n = 2), 3 (n=1) and 5 (n = 1) (Fig. 2). ST94 and CC94 are widely detected in the U.S., and they are the fourth most frequently seen ST and CC in North America. They are related to the pathogenic strains of serotypes 3, 4, 5, 7 and 24, but unrelated to serotypes 1, 2 and 14 43 . ST94 detected in Europe and Asia is mainly related to serotypes 4, 16 and unidentified strains 44 . Taken together, different serotypes exerting distinct infectious efficiency might determine the virulence or geographical distribution of S. suis 45 .
CC1832 is the largest CC in this study. It consisted of ST1831, ST1832, ST1833, ST1836, ST1837, ST1838, ST1840, ST1841 and ST1844, which corresponded mostly to serotypes 7, 8 and 9 (n = 23) and were mostly isolated from the pigs at the nursing stage (n = 23). This CC has a higher proportion of drug resistance to penicillin G, amoxicillin, cefazolin, ceftiofur, tiamulin and enrofloxacin than the other CCs. The pigs at this stage are susceptible to viral infections, such as PRRSV and PCV2, which are likely to cause opportunistic/secondary bacterial respiratory tract infections, e.g., Glaesserella parasuis, Mycoplasma hyorhinis, Bordetella bronchiseptica, and S. suis 46,47 . The antimicrobial agents are frequently used for pigs at this stage, leading to a relatively high drug resistance to common S. suis serotypes. The S. suis isolated from diseased pigs in this study was mostly from the respiratory system, and most of the isolates showed multi-drug resistance. Therefore, clinical use of antimicrobial agents should be selected with caution based on the characteristics of clinical isolates and the results of antimicrobial drug susceptibility.

Conclusion
S. suis is an important bacterial zoonotic pathogen with a high risk of occupational infection. In this study, aside from newly identified STs of S. suis strains, STs and serotypes were found to exert certain association, which can be further related to the feeding stages of pigs and the antimicrobial drug susceptibility. This is the first study that www.nature.com/scientificreports/ reports the serotype distribution, bacterial resistance and molecular epidemiological analysis of S. suis isolated from diseased pigs at different feeding stages in Taiwan. The epidemiological investigation contributes to a better understanding of the role of this bacteria and more proper treatment strategies. www.nature.com/scientificreports/